Polyacrylamide gel electrophoresis (PAGE) is a protein analysis technique which is useful in the separation, identification, approval of purity and determination of size of protein. Particularly, SDS-PAGE (Sodium Dodesyl Sulfate-PAGE) is a method characterized in that protein binds intimately with an anionic surfactant, i. e. SDS, with the ratio of 1.4 to 1, so as to have (-) charge on the surface thereof, and thereby only the size thereof acts as a separation factor. This method is widely used in protein analysis because it can be simply-handled and has a good resolution (see [A.T. Andrews, Electrophoresis, 2nd Ed., Oxford Science Publications, 1-58, (1988)])
Since most of proteins, which are subjected to analysis, are colorless, an appropriate method must be considered for detection. Various detection methods have been reported to the present time such as organic dye staining method, silver staining method, fluorescence staining method and background staining method, etc.
Organic dye staining method is performed by staining protein band with an organic dye such as Amido black 10B, Ponceaus S, Fast green FCF, Coomassie brilliant blue R (abbreviated to CBBR), etc (see [J. of Chromatography A, 698, 123-143 (1995)]). Particularly, since CBBR staining method is relatively simple and inexpensive, it has been generally used. However, it has problems in requiring a long time staining and destaining procedure and not having so good sensitivity. (50 ng on Bovine serum albumin (BSA)).
Silver staining method is performed by adsorbing silver to gels and then, carrying out reduction reaction, which has the highest sensitivity among non-isotopic detection methods. Although its sensitivity reaches to 0.1 ng, it has problems in involving difficult and multiple steps.
Fluorescence staining method is performed by labelling protein with a fluorescent dye. Although it has high detection sensitivity, it has drawbacks in that it involves very complicated steps, and requires the radiation of ultraviolet rays and the use of expensive equipment for quantitation.
Background staining method is performed by forming precipitate on gels except for protein band, the use of which is limited to SDS gels. Although this method is rapid and useful, the resulting band is not persistent and it has difficulty in storing the stained gel. (see [Anal. Biochem. 174, 157-167 (1988)])
Under the above-described situation, it has been required to develop a novel method for detection of protein in a higher sensitivity than the prior method on polyacrylamide gels in a rapid and simple manner.